Peptides containing D-2-alkyl-Tryptophan

ABSTRACT

Peptides containing in its amino acid chain a D-2-alkylTryptophan residue wherein the alkyl group has between one and three carbon atoms and having pharmacological activity equal to or greater than that of analogous peptides containing natural unsubstituted D-Tryptophan residues in place of the D-2-alkylTryptophan. These peptides are more resistant to oxidative degradation which usually takes place, for example, in the presence of reactive radicals or during high energy sterilization than unsubstituted Tryptophan containing peptides.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a division of application Ser. No.08/871,418, filed Jun. 9, 1997, which is a continuation-in-part ofapplication Ser. No. 08/793,922 filed Mar. 6, 1997 now abandoned, acontinuation-in-part of provisional application Ser. No. 60/019,565filed Jun. 11, 1996, which is a continuation-in-part of application Ser.No. 08/530,853 filed Sep. 20, 1995, now U.S. Pat. No. 5,795,957, whichis a continuation-in-part of application Ser. No. 08/016,862 filed Feb.10, 1993, now U.S. Pat. No. 5,668,254, which is a continuation-in-partof application Ser. No. 07/672,300 filed Mar. 20, 1991, abandoned.

TECHNICAL FIELD

The present invention relates to the field of biologically activepeptides. Specifically, this invention relates to biologically activepeptides containing the amino acid D-Tryptophan ("D-Trp").

BACKGROUND ART

It is well known that the incorporation or substitution of aD-Tryptophan residue into a biologically active peptide chain enhancesthe activity of that chain. Furthermore, such incorporation orsubstitution will prolong the biological activity. The prolonged andenhanced effectiveness of such peptides probably relates the increasedresistance to degradation by peptidases.

Examples of D-Tryptophan containing peptides are the LHRH agonists asdescribed by D. H. Coy et al., Journal of Medical Chemistry, volume 19,page 423 (1976), W. Koenig et al., Peptide Chemistry (1987), T. Shibaand S. Sakakibara (eds.), Osaka, Protein Research Foundation, Osaka(1988), page 591, B. J. A. Furr et al., Journal of Endocrinol. Invest.,volume 11, page 535 (1988). Examples of D-Tryptophan containingsomastostatin analogs, such as the peptides octreotide and vapreotideare disclosed by R. Deghenghi, Biomedicine and Pharmacotherapy, volume42, page 585 (1988). Another example of a D-Tryptophan containingpeptide are the synthetic antagonists of Substance P as disclosed by D.Regoli et al., European Journal of Pharmacology, volume 99, page 193,(1984), and GHRP-6 described by C. Y. Bowers et al., Endocrinology,volume 114, page 1537, (1984).

Peptides containing Tryptophan have been subject to degradation due tothe "Kynurenine pathway". In this pathway, the enzyme Tryptophanpyrrolase (i.e., indolamine 2,3-dioxygenase) degrades the pyrrole ringof Tryptophan. Kynurenine and other breakdown products are generated bythis degradation. Some of the breakdown products have been shown to betoxic when present in elevated concentrations as reported by R. M.Silver et al., The New England Journal of Medicine, volume 322, page874, (1990).

D-Tryptophan containing peptides are subject to degradation by oxygenand other reactive radicals as reported by R. Geiger and W. Koenig, "ThePeptides," Academic Press, volume 3, page 82, New York (1981). TheD-Tryptophan in the peptide chain may react with active or activatedgroups when peptides are formulated in certain controlled deliverypharmaceutical compositions, such as those based onpolylactic/polyglycolic acid polymers. Such degradation is thought to befacilitated by either heat or by the presence of catalysts. It is alsopossible that radiolysis products formed during ionizing sterilizationof these pharmaceutical compositions may facilitate the breakdown ofD-Tryptophan. Clearly, the breakdown of D-Tryptophan, and theconcomitant breakdown of the pharmaceutical compound containingD-Tryptophan is an undesirable effect.

Yabe et al., Synthesis and Biological Activity of LHRH AnalogsSubstituted by Alkyl Tryptophans at Position 3, Chem. Pharm. Bul. 27 (8)pp. 1907-1911 (1979) discloses seven analogs of LHRH in which theTryptophan residue at position 3 was replaced by various L-methylTryptophans and L-ethyl Tryptophans. However, each analog testedexhibited reduced hormonal activity compared to synthetic LHRH.

What is needed is a derivative of D-Tryptophan which retains theprolonged and increased biological activity discussed above, whileresisting degradation by indolamine dioxygenase, oxygen or otherreactive radicals. It is of course essential that such a derivative ofD-Tryptophan would maintain biological activity as compared toD-Tryptophan containing bioactive peptides.

One use of such active peptides is for releasing growth hormone ("GH").If sufficiently high GH levels are achieved in mammals after theadministration of compounds capable of inducing such release, growth canbe accelerated, muscular mass can be increased and production of milkcan be enhanced. It is known that the increase of growth hormone levelsin mammals can be achieved by administering growth hormone releaseagents, such as, for example, growth hormone release hormones (GHRH).

The increase of growth hormone levels in mammals can also be obtained byadministering growth hormone release peptides. See, for example, thefollowing U.S. Pat. Nos. 4,223,019, 4,223,020, 4,223,021, 4,224,316,4,226,857, 4,228,155, 4,228,156, 4,228,157, 4,228,158, 4,410,512,4,410,513, 4,411,890 and 4,839,344. Many of the peptides described inthese patents have complex structures, are difficult to synthesize,purify and/or formulate into convenient dosage forms. Additionally, someof these have in vitro activity, but do not exhibit in vivo activity.Further, some of these peptides are not active when administered orally.

One of the more studied growth hormone release peptides is GHRP-6 (C. Y.Bowers et al., Endocrinology 114:1537 (1984) and has the formulaHis-D-Trp-Ala-Trp-D-Phe-Lys-NH₂. GHRP-6 releases growth hormone both invitro and in vivo and is orally active in animals, including humans. Itsmolecular mechanism has been studied, as well as the molecular mechanismof its analogue heptapeptide GHRP-1 (Cheng et al., Endocrinology124:2791 (1989); M. S. Akman et al., Endocrinology 132:1286 (1993)). Itwas found that contrary to natural GHRH, GHRP-1 and GHRP-6 act throughdifferent receptors for the release of GH and also via a differentmechanism, which is independent from cAMP and which operates throughother intracellular pathways, such as through the mobilization ofcalcium supplies and via a proteinkinase C (PKC)-dependent process (L.Bresson-Bepoldin and L. Dufy-Barbe, Cell. Calcium 15, 247 (1994)).

In view of the important effects that growth hormone releasing peptidescan have on veterinary and human medicine, there remains a need forgrowth hormone releasing peptides that are more efficacious than thosecurrently in existence, and as such, can be administered at a lowerconcentration and at a lower cost with fewer adverse health affects.

Therefore, rather simple, short chain oligopeptides capable of promotinggrowth hormone release that can be easily and conveniently prepared andthat can be easily purified and formulated into a dosage form that canbe administered via the oral route are presently desired. In particular,those oligopeptides exhibiting in vivo activity when administered orallyare sought.

The terms "biological effect" or "pharmacological effect" as used in thepresent disclosure refer to the qualitative effect that a bioactivepeptide has upon living tissue. As an example, LHRH, luteinizing hormonereleasing hormone, has the biological effect of causing cells of theanterior pituitary gland to release luteinizing hormone. In contrast,the term "potency" is used in its conventional sense to refer to thedegree and duration of the bioactivity of a given peptide.

Utilizing these terms as defined above, what is needed is a Tryptbphancontaining bioactive peptide which is resistant to oxidative degradationand reactive radical attack while maintaining the same biologicalactivity and a similar or greater potency than the presently availableanalogous peptides provide.

SUMMARY OF INVENTION

The present invention relates to certain defined peptides that contain aD-2-alkylTryptophan ("D-2-Mrp") residue to enhance or increase thebiological activity of the peptide. The alkyl group is substituted atthe number 2 position of the Tryptophan, and typically includes 1 to 3carbons, such as methyl, ethyl, propyl or isopropyl, and is preferably amethyl group.

When known, biologically active peptides that contain Tryptophan aremodified to replace the tryptophan with a D-2-alkyl Tryptophan, improvedresistance to oxidative breakdown is achieved while maintaining orenhancing the biological activity of the peptide.

These peptides generally have a sequence of two to ten amino acids,wherein at least one amino acid is the D-2-alkylTryptophan. In somepeptides, at least two amino acids are D-2-alkylTryptophan, in adjacentpositions in the sequence.

One embodiment of the invention relates to peptides which enhance therelease of growth hormone in vivo. These peptides have one of thefollowing formulae:

    A--D--X--Z--B                                              (I)

    E--D--Mrp--(Ala).sub.n --F--G                              (II)

    J--D--X--Mrp--NH.sub.2                                     (III)

wherein

A is hydrogen, 2-aminoisobutyryl, or 4-aminobutyryl;

D stands for the dextro isomer,

X is a 2-alkyltryptophan of formula (IV): ##STR1## wherein R ishydrogen, CHO, SO₂ CH₃, mesitylene-2-sulfonyl, PO₃ (CH₃)₂, PO₃ H₂,wherein R₁ is a C₁ -C₃ alkyl group (e.g., methyl, ethyl, propyl orisopropyl), or X is a residue of protected serine, Ser (Y), wherein Ycan be benzyl, p-chlorobenzyl, 4-methoxybenzyl, 2,4,6-trimethoxybenzyl,or t-butyl,

B is NR₂ R₃, wherein R₂ and R₃, which can be the same or different, arehydrogen, a C₁ -C₃ alkyl group, an OR₄ group, wherein R₄ is hydrogen, aC₁ -C₃ alkyl, or a C-Lys-NH₂ group, wherein C is Phe, Mrp or D-Mrp;

Z is D-Mrp, D-βNal or Mrp;

E is any natural L-amino acid or its D-isomer, imidazolylacetyl,isonipecotinyl, 4-aminobutyryl, 4-(aminomethyl) cyclohexanecarbonyl,Glu-Tyr-Ala-His, Tyr-Ala-His, Tyr-His, D-Thr-His, D-Ala, D-Thr, Tyr andGly;

n is 0 or 1;

F is selected from the group consisting of Trp, D-Trp, Phe and D-β-Nal;

G is selected from the group consisting of NH₂, D-Phe-Lys-NH₂,Phe-Lys-NH₂, D-Trp-Lys-NH₂, D-Phe-Lys-Thr-NH₂, D-Phe-Lys-D-Thr-NH₂ andan O-C₁ -C₃ alkyl group, with the proviso that E is not His when F isL-Trp or D-Trp and when G is D-Phe-Lys-NH₂ ; and

J is hydrogen, 4-aminobutyryl (or GAB) or D-Mrp.

Additional peptides are disclosed which are useful for enhancing theactivity of hormones acting on the hypothalamic pituitary axis. Thesepeptides have the following formula:

    K--D--2--Mrp--M (V)

wherein

K is Ala-His-, D-Phe-Cys-Phe-, D-Phe-Cys-Tyr-, Arg-D-Trp-N-methyl-Phe-,D-pyro-Gln-Gln-D-Trp-Phe-, or pyro-Glu-His-Trp-Ser-Tyr-; and

M is Met-NH₂, Leu-Met-NH₂, Leu-Arg-Pro-NH₂, Leu-Arg-Pro-Gly-NH₂,Ala-Trp-D-Phe-Lys-NH₂, Lys-Val-Cys-Trp-NH₂, or Lys-Thr-Cys-NHCH(CH₂OH)CHOHCH₃.

Preferred peptides have the following formula:

    P--D--Mrp--(D--Q).sub.n --T                                (VI)

wherein

P is hydrogen, 2-aminoisobutyryl, 4-aminobutyryl, imidazolylacetyl,isonipecotinyl, or 4-(aminomethyl)-cyclohexanecarbonyl (or tranexamyl);

D stands for the dextro isomer;

Mrp is a 2-alkyltryptophan of formula (IV);

Q is Trp, Mrp, or β-Nal;

n is 0, 1 or 2; and

T is OCH₂ CH₅, NH₂, Mrp-NH₂, Mrp-Lys-NH₂, Lys-NH₂, or Phe-Lys-NH₂ ;

with the proviso that, when P is 4-aminobutyryl or4-(aminomethyl)-cyclohexanecarbonyl, Q is not β-Nal; and when P is H or4-aminobutyryl, n is not 1 when T is NH₂.

Any pharmaceutically acceptable salt of these peptides can also be used.Addition salts with pharmaceutically acceptable organic or inorganicacids are preferred.

Another embodiment of the invention relates to a method for enhancingthe biological activity and oxidation resistance of a peptide byformulating a composition comprising one of the peptides described aboveand administering a therapeutically effective amount of the compositionto an animal. Advantageously, the animal is a human, and the peptide isadministered in an amount of about 0.1 μg to about 10 μg of totalpeptide per kg of body weight.

The pharmaceutical composition which contain a therapeutically effectiveamount of one of these peptides, optionally in admixture with a carrieror an excipient, form another embodiment of the invention. Thesecompositions can be provided in the form of a composition forparenteral, intranasal, oral, or controlled release administration, oras a subcutaneous implant, where the peptide is administered orally inan amount of about 30 μg to about 1000 μg of peptide per kg of bodyweight. The peptide is typically in the form of a pharmaceuticallyacceptable addition salt, and the carrier can be a biodegradable polymermatrix so that the composition is in a controlled release dosage form.Implants are conveniently used for this purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the stability of D-Trp andD-2-methyl-Trp in an acid solution;

FIG. 2 is a graphical representation of the oxidative degradation ofD-Trp and D-2-methyl-Trp in solution;

FIGS. 3-5 are graphical representations of GH release in anesthetizedmale rats following intravenous administration of saline, GHRP-6 andHEXARELIN;

FIGS. 6-11 are graphical representations of GH release in anesthetizedmale rats following subcutaneous administration of saline, GHRP-6 andHEXARELIN;

FIG. 12 is a graphical comparison of the hydrophobicity of GHRP-6 andHEXARELIN;

FIG. 13 is a graphical representation of the effect of irradiation onGHRP-6 and HEXARELIN in an acetate buffer solution;

FIGS. 14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b are graphicalrepresentations of GH release in anesthetized male rats followingintravenous administration of saline, GHRP-6 and HEXARELIN;

FIGS. 18-20 are graphical representations of the effect of HEXARELIN ongrowth hormone secretion in young healthy male volunteers;

FIGS. 21A and 21B are graphs of the amount of growth hormone andcortisol released in humans after administration of the peptide ofExample 17; and

FIGS. 22-24 are graphs of the amount of growth hormone released in dogsafter oral administration of various amounts of the peptideGAB-D-2-Mrp-D-βNal-Phe-Lys-NH₂ (FIG. 1) and Aib-D-2-Mrp-D-2-Mrp-NH₂(FIGS. 2 and 3).

DETAILED DESCRIPTION OF THE INVENTION

Now in accordance with the present invention, a derivative ofD-Tryptophan has been discovered which enhances or increases thebiological activity of certain peptides, or which imparts tobiologically active peptides incorporating that derivative improvedresistance to the oxidative breakdown reaction of Tryptophan, whilemaintaining or increasing the biological activity and pharmacologicaleffect compared to peptides incorporating unaltered D-Tryptophan.

Specifically, the present invention relates to a Tryptophan derivative,namely D-2-Mrp, in which the alkyl substituent in the 2 position is alower alkyl group, preferably one containing 1 to 3 carbon atoms.Peptides incorporating D-2-Mrp are more stable in the presence ofreactive radicals or when pharmaceuticals containing such peptides areexposed to ionizing radiation.

This invention also describes a more practical synthesis of D-2-Mrp andthe preparation of novel protected D-2-Mrp derivatives particularlysuited for use in the synthesis of peptides.

A particularly preferred biologically active peptide containing thismodified Tryptophan derivative is an analog of GHRP (Growth HormoneReleasing Peptide), His-D-2-alkyl-Trp-Ala-Trp-D-Phe-Lys-NH₂, which isreferred to in the trade as HEXARELIN. Additional biologically activepeptides include:

Ala-His-D-2-alkyl-Trp-Ala-Trp-D-Phe-Lys-NH₂,

Pyro-Glu-His-Trp-Ser-Tyr-D-2-alkyl-Trp-Leu-Arg-Pro-Gly-NH₂,

Pyro-Glu-His-Trp-Ser-Tyr-D-2-alkyl-Trp-Leu-Arg-Pro-NHCH₂ CH₃,

D-Pro-Gln-Gln-D-Trp-Phe-D-Trp-2-alkyl-Trp-Met-NH₂,

Arg-D-Trp-N-methyl-Phe-D-2-alkyl-Trp-Leu-Met-NH₂,

D-Phe-Cys-Phe-D-2-alkyl-Trp-Lys-Thr-Cys-NHCH (CH₂ OH) CHOHCH₃ and

D-Phe-Cys-Tyr-D-2-alkyl-Trp-Lys-Val-Cys-Trp-NH₂

where alkyl designates a lower alkyl group, preferably comprising 1 to 3carbons. The methyl group is most preferred due to simplicity ofmanufacture.

The first peptide mentioned above, named HEXARELIN, is an analog of GHRpand is used for stimulating the release of growth hormone. The secondpeptide is an analog of the first and contains one additional aminoacid.

The third and fourth peptides listed above are analogs of the naturalpeptide Pyro-Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-NH₂, which is aluteinizing hormone releasing hormone (LH-RH), i.e., a neurohumoralhormone produced in the hypothalamus which stimulates the secretion ofthe LH luteinizing hormone by the anterior pituitary gland. Thesepeptides pertain therefore to the class of LHRH agonists and are alsodefined respectively as follows:

D-2-methyl-Trp⁶ !LHRH and Des-Gly¹⁰ -D-2-methyl-Trp⁶ -Pro-ethylamide⁹!LHRH.

The fifth and sixth peptides listed above are antagonists of substanceP. Substance P is a neurotransmitter used by sensory neurons that conveyresponses of pain or other noxious stimuli to the central nervoussystem. Accordingly, these peptides have analgesic and anti-inflammatoryeffects.

The seventh and eighth peptides are analogs (agonists) of somatostatinand as such show antisecretory and antitumoral activity.

Although the aforementioned examples of the present invention disclosespecific embodiments thereof, it is believed that the substitution of anD-2-Mrp in bioactive peptide containing at least one Tryptophan residuewill yield bioactive peptides providing the advantages and benefitsdiscussed above.

The incorporation of a D-2-Mrp in bioactive peptides as described aboveprovides a method for prolonging and preserving the activity of suchpeptides. When analogous bioactive peptides not substituted with aD-2-Mrp are exposed to various processing conditions and substances, theactivity of such peptides may be adversely effected. Sterilizingprocedures used in the pharmaceutical industry may expose bioactivecompounds to ionizing radiation. Such radiation may effect the formationof reactive radicals. Tryptophan containing peptides are particularlysusceptible to attack by such radicals and such attack may render thepeptide ineffective, or possibly toxic. Furthermore, various formulatingcompounds, such as polylactic-polyglycolic acid polymers may containactive, or activated groups which may also attack Tryptophan containingbioactive peptides. The present invention provides a method forprotecting a tryptophan containing bioactive peptide from thesemanufacturing hazards while also increasing the peptides resistance tooxidative degradation after formulation is complete. It is believed thatthe presence of the alkyl group at the number 2 position of theTryptophan increases the stability of the pyrrole ring wherein attack byreactive radicals and active or activated groups occurs.

2-methyl-Tryptophan is known (cf. H. N. Rydon, J. Chem. Soc. 1948, 705)and the homologous alkylated derivatives are conveniently prepared fromthe corresponding 2-alkyl indoles by well known methods (cf. J. P. Li etal., Synthesis (1), 73, 1988). The resolution of the racemic Tryptophanderivatives to give the D-enantiomers of the present invention can beachieved by a variety of methods (cf. Amino Acids, Peptides andProteins, Vol. 16, pages 18-20, The Royal Society of Chemistry, London,1985). Specifically, S. Majima (Hoppe-Seyler's Z. Physiol. Chem. 243,250 (1936) describes the digestion of 2-methyl tryptophan withcolibacteria with isolation of the undigested D-isomer.

A more practical synthesis of D-2-methy tryptophan is presented inExample 1. In general, D-2-Mrp can be prepared by a method whichcomprises treating a solution of racemic N.sup.α-acetyl-2-alkyl-Tryptophan with acylase for a sufficient time and at asufficient temperature to form insoluble material therein, recoveringand lyophilizing the insoluble fraction to form a residue, dissolvingthe residue in a suitable solvent, subjecting the solvent and dissolvedresidue to chromatography to obtain highly polar fractions and lesserpolar fractions, collecting the lesser polar fractions to obtain aN.sup.α -acetyl-D-2-alkyl-Tryptophan compound and hydrolyzing thiscompound to obtain D-2-Mrp.

In this method, the racemic Nα-acetyl-2-alkyl-Tryptophan is treated bydissolution in water with a base, such as potassium hydroxide, andretaining the solution for about 24 hours at about 40° C. TheNα-acetyl-D-2-alkyl-Tryptophan is then hydrolyzed under an inert gas,such as nitrogen, with a base, such as KOH or NaOH, for about 24 hoursat 100° F., prior to the addition of an acid, such as acetic acid, andthe cooling of the solution. Also, the insoluble fraction can beobtained by filtration and the residue may be formed by lyophilizing theinsoluble fraction to dryness. It is preferred for the residue to bedissolved in the upper phase of N-BaOH-AcOH-H₂ O before being introducedinto the chromatography column.

Both the solution phase or the solid phase method of peptide synthesiscan be used to make the peptides of this invention, (cf. R. Geiger etal., "The Peptides", Academic Press, New York 1981). If the solid phasemethod is used, peptide synthesizers such as the Applied Biosystem 430A,Bioresearch Sam 9500 or the Beckman Model 990 are preferably used.According to this methodology, the first amino acid is linked to thebenzhydrylamine resin and the remaining protected amino acids are thencoupled in a step wise manner using the standard procedures recommendedby the manufacturers of the synthesizers. For instance, amino acidcouplings are performed by using symmetrical anhydrides in the AppliedBiosystems Synthesizer and diisopropylcarbodiimide in the Bioresearch orBeckman machines. The amino acid derivatives are protected by thetertiary butoxy-carbonyl groups or by Fmoc (9-Fluorenyl methoxycarbonyl)groups on the alpha-amino function during the synthesis. The functionalgroups present in the amino-acid in the side chain are previouslyprotected, e.g. by acetyl(Ac), benzoyl (Bz), t-butyloxycarbonyl (Boc),benzyloxymethyl (Bom), benzyl (Bzl), benzyloxycarbonyl (Z), formyl(For), p-nitro-phenyl ester (ONp), tosyl (Tos), etc. For instance, thefunctional groups of Histidine are protected by benzyloxymethyl(His(Bom)), tosyl (His(Tos)), the functional groups of Tryptophan byformyl (Trp(For)), those of Serine by benzyl (Ser(Bzl)), those ofTyrosine by 2-Br-benzyloxycarbonyl (Tyr(2-Br-Z)), those of Arginine bytosyl (Arg(Tos)), those of Leucine by O-benzyl-p-tosyl(Leu(O-Bzl-p-Tos)), those of Proline by O-benzyl HCl (Pro(O-Bzl HCl)),those of Glycine by O-benzyl HCl (Gly (O-Bzl HCl)), those of Cysteine by4-methyl-benzyl (Cys(4-Me-Bzl)), those of Lysine by benzyloxycarbonyl(Lys(Z)), those of Threonine by benzyl-OH (Thr(Bzl-OH)), those of Valineby O-benzyl-tosyl (Val(O-Bzl-p-Tos)), those of Glutamic Acid by O-benzyl(Glu(O-Bzl)), those of Methionine by P-nitrophenyl ester (Me(Onp)), andthose of Alanine by O-benzyl HCl (Ala(O-Bzl HCl).

The Boc protective groups on the alpha-aminic function are removed ateach stage by treatment with 60% trifluoroacetic acid ("TFA") indichloromethane. Cleavage of Trp and Met containing peptides from theresin with simultaneous removal of all side-chain protecting groups isachieved as described by J. P. Tam et al., J. Am. Chem. Soc., Vol 105,page 6442 (1983). The crude peptides after HF cleavage are purified on aSephadex G-50 F column in 50% acetic acid or by preparative reversephase HPLC using gradients of acetonitrile and water containing 0.1%trifluoroacetic acid.

Another embodiment of the present invention relates to a number ofshort-chain oligopeptides which promote the release and increase ofgrowth hormone levels in blood of animals by including in the peptidechain a D-Mrp residue. In a completely surprising manner it has now beenfound that very short oligopeptides, having at least one D-2-Mrpresidue, have activity releasing growth hormone (GH) from somatotropes.Another unexpected distinctive feature of the present invention is thevery high potency and the favorable oral activity/oral potency ratiothat even the smallest tripeptides of the series exhibit.

It has also been found that the introduction of a D-2-Mrp residue inoligopeptides of the GHRP series, modifies the intracellular mechanismof GH release. In addition, the introduction of D-2-Mrp into a GHRPresults in a substantial increase of the activity of the adenylcyclasein the anterior pituitary glands, both of murine origin, and of humanorigin.

Another unexpected characteristic feature of the present invention isthe very high potency of penta-, hexa-, and heptapeptides and thefavorable oral activity/potency ratio of shorter-chained tri- andtetrapeptides of the series.

Short chain oligopeptides within the scope of the present invention aredefined by the following formulae:

    A--D--X--Z--B (I)

    E--D--Mrp--(Ala).sub.n --F--G                              (II)

    J--D--X--Mrp--NH.sub.2                                     (III)

wherein

A is hydrogen, 2-aminoisobutyryl (i.e., alpha-aminoisobutyric acid), or4-aminobutyryl (i.e. gamma-aminoisobutyric acid);

D relates to the dextro isomer,

X is Mrp, i.e., a 2-alkyltryptophan of formula (IV): ##STR2## wherein Ris hydrogen, CHO, SO₂ CH₃, mesitylene-2-sulfonyl, PO₃ (CH₃)₂, PO₃ H₂,wherein R₁ is a C₁ -C₃ alkyl group (e.g., methyl, ethyl, propyl orisopropyl), or X is a residue of protected serine, Ser (Y), wherein Ycan be benzyl, p-chlorobenzyl, 4-methoxybenzyl, 2,4,6-trimethoxybenzyl,or t-butyl,

B is NR₂ R₃, wherein R₂ and R₃, which can be the same or different, arehydrogen, a C₁ -C₃ alkyl group, an OR₄ group, wherein R₄ is hydrogen, aC₁ -C₃ alkyl, or a C-Lys-NH₂ group, wherein C is Phe, Mrp or D-Mrp;

Z is D-Mrp, D-βNal or Mrp;

E is any natural L-amino acid or its D-isomer, imidazolylacetyl,isonipecotinyl, 4-aminobutyryl, 4-(aminomethyl)cyclohexanecarbonyl,Glu-Tyr-Ala-His, Tyr-Ala-His, Tyr-His, D-Thr-His, D-Ala, D-Thr, Tyr andGly;

n is 0 or 1;

F is selected from the group consisting of Trp, D-Trp, Phe and D-β-Nal;

G is selected from the group consisting of NH₂, D-Phe-Lys-NH₂,Phe-Lys-NH₂, D-Trp-Lys-NH₂, D-Phe-Lys-Thr-NH₂, D-Phe-Lys-D-Thr-NH₂ andan O--C₁ -C₃ alkyl group, with the proviso that E is not His when F isL-Trp or D-Trp and when G is D-Phe-Lys-NH₂ ; and

J is hydrogen, GAB or D-Mrp.

Also included are any addition salts of any pharmaceutically acceptableorganic or inorganic acids of any one of these short chainoligopeptides.

The abbreviations for the residues of amino acids herein used are inagreement with the standard nomenclature for the peptides: e.g.,

Gly=Glycine

Tyr=L-Tyrosine

Ile=L-Isoleucine

Glu=L-Glutamic Acid

Thr=L-Threonine

Phe=L-Phenylalanine

Ala=L-Alanine

Lys=L-Lysine

Asp=L-Aspartic Acid

Cys=L-Cysteine

Arg=L-Arginine

Gln=L-Glutamine

Pro=L-Proline

Leu=L-Leucine

Met=L-Methionine

Ser=L-Serine

Asn=L-Asparagine

His=L-Histidine

Trp=L-Tryptophan

Val=L-Valine

D-β-Nal=D-9-Naphthylalanine

Moreover, the following abbreviations are also used:

Aib=2-aminoisobutyryl;

GAB=4-aminobutyryl;

INIP=Isonipecotinyl

IMA=Imidazolylacetyl

Mrp=2-alkyltryptophan

Bzl=benzyl;

p-Cl-Bzl=p-chlorobenzyl;

Mob=4-methoxybenzyl;

Tmob=2,4,6-trimethoxybenzyl;

tbu=tert-butyl;

For=formyl;

Mts=mesitylene-2-sulfonyl.

According to the present invention, alkyl means lower alkyl, comprisingfrom 1 to 3 carbon atoms. Examples of lower alkyl are methyl, ethyl,propyl, isopropyl. Among these, the methyl group is most preferred.

All the three letter-abbreviations of the amino acids preceded by a "D"indicate the D-configuration of the amino acid residue. When the aminoacid is referred to with the only three-letter abbreviation, it has Lconfiguration.

In addition, the peptides of the present invention can bear, on theC-terminus thereof, a C₁ -C₃ alkyl ester, wherein alkyl is selected fromthe group consisting of methyl, ethyl, n-propyl and iso-propyl.

As used herein, "natural L-amino acid" refers to an amino acid bearingthe L-configuration and being found in nature. Examples of naturalL-amino acids include, but are not limited to L-tyrosine, L-isoleucine,L-glutamic acid, L-threonine, L-phenylalanine, L-alanine, L-lysine,L-aspartic acid, L-cysteine, L-arginine, L-glutamine, L-proline,L-leucine, L-methionine, L-serine, L-asparagine, L-histidine,L-tryptophan and L-valine.

As used herein, "therapeutically effective" means an amount or dosewhich, when administered to the animal including human, patient orsubject, renders a benefit or an effect of increasing the level ofcellular proteins such as hormones, or renders a benefit or an effect oftreating or preventing an abnormal biological condition or disease.

As used herein, "EC₅₀ " refers to the effective concentration for 50% ofthe peptides.

The most preferred growth hormone-release peptides of the presentinvention are:

GAB-D-Mrp-D-Mrp-Phe-Lys-NH₂ ;

GAB-D-Mrp-D-Mrp-Mrp-Lys-NH₂ ;

Aib-D-Mrp-D-Mrp-NH₂ ;

Aib-D-Mrp-Mrp-NH₂ ;

Aib-D-Ser(Bzl)-D-Mrp-NH2; and

GAB-D-Mrp-D-βNal-Phe-Lys-NH₂

INIP-D-Mrp-D-Trp-Phe-Lys-NH₂ ;

INIP-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ;

IMA-D-Mrp-D-Trp-Phe-Lys-NH₂ ;

IMA-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ;

GAB-D-Mrp-D-Trp-Phe-Lys-NH₂ ;

GAB-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ;

GAB-D-Mrp-D-β-Nal-NH₂ ;

GAB-D-Mrp-D-β-Nal-OC₂ H₅ ;

imidazolylacetyl-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂ ;

imidazolylacetyl-D-Mrp-D-Trp-Phe-Lys-NH₂ ;

imidazolylacetyl-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ;

4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-Trp-Phe-Lys-NH₂ ;

4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ;

D-Ala-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂ ;

D-Thr-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂ ;

His-D-Mrp-Ala-Phe-D-Trp-Lys-NH₂ ;

His-D-Mrp-Ala-Trp-D-Phe-Lys-Thr-NH₂ ;

His-D-Mrp-Ala-Trp-D-Phe-Lys-D-Thr-NH₂ ;

Tyr-His-D-Mrp-Ala-Trp-D-Phe-LysNH₂ ;

His-D-Mrp-Ala-TrpNH₂ ;

D-Thr-His-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂ ; and

D-Thr-D-Mrp-Ala-TrpNH₂.

Pharmaceutically acceptable salts of the oligopeptide compounds of thepresent invention include but are not limited to organic or inorganicaddition salts such as for example hydrochloride, hydrobromide,phosphate, sulfate, acetate, succinate, ascorbate, tartrate, gluconate,benzoate, malate and fumarate salts.

These peptides are preferably administered by the oral route, but theyalso can be administered intranasally, buccally or parenterally. Thesecompounds (i.e., oligopeptides of the present invention) can beformulated into controlled release dosage forms, such as, biodegradablemicrocapsules, microspheres, subcutaneous implants and the like. Othercontrolled release dosage forms, though not specifically listed, areknown to those skilled in the art and are within the scope of thepresent invention.

The peptides according to the present invention can be synthesizedaccording to the usual methods of peptide chemistry, both solid-phaseand solution, or by means of the classical methods known in the art. Thesolid-phase synthesis starts from the C-terminal end of peptide. Asuitable starting material can be prepared, for example, by attachingthe required protected alpha-amino acid to a chloromethylated resin, ahydroxymethylated resin, a benzylhydrylamine resin (BHA), or to aparamethylbenzylhydrylamine resin (p-Me-BHA). More particularly, forexample, a chloromethylated resin is sold with the Trade Mark BIOBEADS(R) SX 1 by BioRad Laboratories, Richmond, Calif. The preparation of thehydroxymethyl resin is described by Bodansky et al., Chem. Ind. (London)38, 15997, (1966). The BHA resin is described by Pietta and Marshall,Chem. Comm., 650 (1970) and is commercially available from PeninsulaLaboratories Inc., Belmont, Calif.

After the starting attachment, the alpha-amino acid-protecting group canbe removed by means of different acid reagents, comprisingtrifluoroacetic acid (TFA) or hydrochloric acid (HCl) dissolved inorganic solvents at room temperature. After the removal of thealpha-amino acid-protecting group, the remaining protected amino acidscan be coupled step by step in the desired order. Each protected aminoacid can generally be reacted in excess of about three times using asuitable carboxyl activating group, such as dicyclohexylcarbodiimide(DCC) or diisopropylcarbodiimide (DIC) dissolved, for example, inmethylene chloride (CH₂ Cl₂) or dimethylformamide (DMF) and mixturesthereof. After the desired amino acid sequence has been completed, thedesired peptide can be cleaved from the supporting resin by treatmentwith a reagent such as hydrogen fluoride (HF), which not only cleavesthe peptide from the resin, but also the more common protecting groupsof the lateral chains. When a chloromethylated resin or ahydroxymethylated resin is used, the treatment with HF leads to theformation of the acid peptide in free form. When a BHA or p-Me-BHA resinis used, the treatment with HF directly leads to the formation of theamide peptide in free form.

The above discussed sold-phase procedure is known in the art and wasdescribed by Atherton and Sheppard, Solid Phase Peptide Synthesis (IRLPress, Oxford, 1989).

Some methods in solution, which can be used to synthesize the peptidemoieties of the present invention are detailed in Bodansky et al.,Peptide Synthesis, 2nd edition, John Wiley & Sons, New York, N.Y. 1976and from Jones, The Chemical Synthesis of Peptides, (Clarendon Press,Oxford, 1994).

These compounds can be administered to animals and humans at aneffective dose which can be easily determined by one of ordinary skillin the art and which can vary according to the specie, age, sex andwight of the subject to be treated. For example, in humans, whenintravenously administered, the preferred dose falls in the range fromabout 0.1 μg to about 10 μg of total peptide per kg of body weight. Whenorally administered, typically higher amounts are necessary. Forexample, in humans for the oral administration, the dosage level istypically from about 30 μg to about 1000 μg of peptide per kg of bodyweight. The exact level can be easily determined empirically by theskilled artisan.

Compositions useful for releasing growth hormone in an animal, includinga human, can comprise a peptide of the present invention or apharmaceutically acceptable salt thereof, or combinations of peptides ofthe present invention or pharmaceutically acceptable salts thereof,optionally, in admixture with a carrier excipient, vehicle, diluent,matrix or delayed release coating. Examples of such carriers,excipients, vehicles and diluents, can be found in Remington'sPharmaceutical Sciences, Eighteenth Edition, A. R. Gennaro, Ed., MackPublishing Company, Easton, Pa., 1990. The delayed releasepharmaceutical forms, comprising bioerodible matrixes suitable forsubcutaneous implant are particularly useful. Examples of these matricesare described in WO 92/22600 and WO 95/12629.

The following examples are only provided as being illustrative ofpreferred embodiments of the present invention and are not intended tolimit the breadth and scope of the invention as is readily understood bythose skilled in the art.

EXAMPLES

The following examples illusrtate the method of making and activity ofthe peptides which represent preferred embodiments of the invention:

Example 1

Synthesis of D-2-Methyl-Tryptophan

N⁶⁰ -Acetyl-2-methyl-D,L-tryptphan Y. Yabe et al. Chem. Pharm. Bull27(8) 1907-1911 (1979)! 1.3 g (5 mmol), was suspended in 50 ml of waterand dissolved by adding concentrated ammonium hydroxide to a pH of 7.5.5 mg of acylase (from porcine kidney, Sigma Grade III lyophilized) wasadded and the mixture kept at 40° C. for 24 hours. The insolublematerial was separated by filtration and the filtrate was lyophilized todryness. The residue was dissolved in the upper phase (10 ml) ofn-BuOH-AcOH-H₂ O (16:1:20) and chromatographed on a 3.5×50 cm column ofSephadex G-25 (Pharmacia, Fluka) collecting 10 ml fractions. The lesspolar fractions (N° 16-25) were pooled to give mainly undigested N.sup.α-acetyl-2-methyl-D-tryptophan which, without purification, washydrolyzed under N₂ with a solution of 1 g KOH in 25 ml of water at 100°F. for 24 hours. Acetic acid (2 ml) and water (10 ml) were added to thehot solution and placed in the refrigerator for 12 hours. The cruderesulting 2-methyl-D-tryptophan was filtered, washed and dried andrecrystallized from hot water (charcoal) to yield the title compound,m.p. 244-246°, α!_(D) ²⁰ +18.6, cO.26(H₂ O)!.

The enhanced stability of the 2-methyl-Tryptophan derivative isillustrated in FIGS. 1 and 2. In FIG. 1, the stability of thisderivative is compared to D-Trp for 1% solutions at a pH of 2.2 (0.2 Mcitrate buffer with 0.02% NaN₃ added) which are maintained in the darkunder helium, while FIG. 2 shows the oxidative degradation of thesecompounds in 1% solutions at a pH of 5.4 (0.2M acetate buffer with 0.02%NaN₃ added) under oxygen and constant light. The peak area is measuredby HPLC.

The results show that the substituted Trp is stable for 60 days orlonger, whereas the unsubstituted Trp began to lose stability afterabout 20 days.

Example 1a

Synthesis of Fmoc-D-2-Methyl-Trp

N.sup.α - 9 Fluorenylmethyloxycarbonyl!-2-methyl-D-Tryptophan (Fmocderivative)

To a suspension of 436 mg of 2-methyl-D-Tryptophan of Example 1 (2mMole) in 5 ml of water, a solution of 710 mg (2.1 mMole) of Fmoc-OSu (9FluorenylmethyloxyN-hydroxysuccinimide) in 2 ml of dioxane is addeddropwise and the mixture is stirred overnight at room temperature. Themixture is extracted with ether and the ether phase discarded. Theaqueous phase is adjusted to pH 1 with 6N HCl and extracted withethylacetate. The organic phase is washed with water, dried over Na₂ SO₄and evaporated in vacuo. The residue is dissolved in ether and hexane isadded to precipitate the crystalline product which is filtered anddried.

M.p. 196-198° C. TLC: Rf 0.45 in CHCl₃ /MeOH/AcOH85/10/5

    ______________________________________            C          H       N    ______________________________________    Calculated              73.62%       5.49%   6.36%    Found:    73.72%       5.29%   6.27%    ______________________________________

Additional suitably protected 2-Methyl-D-Tryptophans have the formula:##STR3## Where X is BZ (Benzoyl) or Z (Benzyloxycarbonyl). These can beprepared by conventional methods, starting from the2-Methyl-D-Tryptophan.

Examples 2-9

Peptides which include the D-2-methyl Trp were made as follows:

Example 2

Pyro-Glu-His-Trp-Ser-Tyr-D-2-alkyl-Trp-Leu-Arg-Pro-Gly-NH₂,

The protective groups for the side chains are Tosyl (Tos) for Arginineand Histidine and Bromo-benzyloxycarbonyl (2-Br-Z) for Tyrosine. Thebenzhydrylamine resin (2.2 g) (Bachem®), was cross-linked at 1% withProline and the apparatus used was a Beckman Model 990. The amino acidsprotected by Boc (tert-butyloxycarbonyl) are coupled withdicyclohexylcarbodiimide. The Boc groups are removed by trifluoroaceticacid in methylene chloride.

The synthesis yielded 4.07 g of the decapeptide-resin (98% oftheoretical weight gain). Part of this resin (1.5 g) was stirred at 0°centigrade for 30 minutes with HF (24 ml) and anisole (8 ml). HF wasthen removed as rapidly as possible (ca. 60 min) in vacuo and EtOAc wasadded to the thus obtained residue. Solid material was filtered, washedwith EtOAc, dried, and extracted with 2 M AcOH. Lyophilization gave awhite powder which was purified by gel filtration on a column (2.5×95cm) of Sephadex G-25 (fine) by elution with 2 M ACOH. The eluate portioncorresponding to the major peak was then dried and eluted further on acolumn (2.5×95 cm) of Sephadex G-25 (fine) previously equilibrated withthe lower phase followed by the upper phase of the following biphasicsolvent mixture n-BuOH-AcOH-H₂ O (4:1:5). Elution with the upper phasegave a major peak and the peptide from this area was collected,concentrated to dryness, and lyophilized from dilute AcOH to give thetitled peptide as a white powder. Amino acid analysis was consistentwith the desired structure.

Example 3

Pyro-Glu-His-Trp-Ser-Tyr-D-2-alkyl-Trp-Leu-Arg-Pro-NHCH₂ CH₃,

The peptide was assembled on a 1% cross-linked Pro-Merrifield resin (2.0g, 1.0 mmol of Pro) using the same conditions and protecting groupsemployed in Example 1, with the exception that dinitrophenol groupprotection was used for the imidazole group of histidine. Thepeptide-resin obtained (3.45 g) was stirred with ethylamine (20 ml, 0°C.) for 6 hours and excess amine was removed in vacuo. The protectedpeptide resin was extracted with MeOH and precipitated by the additionof a large excess of EtOAc to give 1.36 g of material. The obtainedproduct was treated and deprotected with HF-anisole and crude peptideobtained after this treatment was purified by gel filtration followed bypartition chromatography to yield the homogeneous peptide cited. Aminoacid analysis was consistent with the desired structure.

Examples 4-9

Using the above described methods with appropriate modifications wellknown to the skilled in the art particularly the use of Fmoc derivativesas protected amino acids of Example 1a for the preparation of Fmoc-D-2Methyl Trp or other suitably protected amino acids, the followingpeptides are synthesized:

Example 4

His-D-2-alkyl-Trp-Ala-Trp-D-Phe-Lys-NH₂ ("HEXARELIN"),

Example 5

Ala-His-D-2-alkyl-Trp-Ala-Trp-D-Phe-Lys-NH₂,

Example 6

D-Pro-Gln-Gln-D-Trp-Phe-D-Trp-2-alkyl-Trp-Met-NH₂,

Example 7

Arg-D-Trp-N-methyl-Phe-D-2-alkyl-Trp-Leu-Met-NH₂,

Example 8

D-Phe-Cys-Phe-D-2-alkyl-Trp-Lys-Thr-Cys-NHCH(CH₂ OH) CHOHCH₃ and

Example 9

D-Phe-Cys-Tyr-D-2-alkyl-Trp-Lys-Val-Cys-Trp-NH₂.

The peptides of Examples 4 and 5 were tested as Growth Hormone releasersin rats. GH released in a series of seven 10-day old rats, injectedsubcutaneously with a standard dose of 160 μg/kg and sacrificed 15minutes after the injection. Results are as follows:

    ______________________________________    Samples         GH (ng/kg)    ______________________________________    Untreated Controls                     14.64 + 21.41    Example 4       201.00 + 39.55    Example 5       212.00 + 48.63    ______________________________________

Thus, the peptides of Examples 4 and 5 (i.e.,His-D-2-methyl-Trp-Ala-Trp-D-Phe-Lys-NH₂ andAla-His-D-2-methyl-Trp-Ala-Trp-D-Phe-Lys-NH₂) were found to be veryactive analogs.

Example 10

The peptide of Example 4 (i.e., HEXARELIN) was compared with GHRP-6{Bowers C. Y., Momany F. A., Reynolds G. A., and Hong A. (1984) On thein Vitro and in Vivo Activity of a New Synthetic Hexapeptide that Actson the Pituitary to Specifically Release Growth Hormone, Endocrinology,114: 1537-1545} both in vitro and in vivo, as follows.

Male Sprague Dawley rats (Charles River, Calco, Italy) of 2-3 months ofage were used. Rats were housed at 22 +2° C., with lighting cycle of 14h light: 10 h dark (lights on from 06.00 to 20.00 h), at least 10 daysbefore starting the experiments. A standard dry diet and water wereavailable ad libitum.

In Vitro Experiments

Pituitary Cell Culture

Pituitary tissue used for cell dissociation included only the anteriorlobe. Briefly, pituitary glands were collected in sterile F-10 mediumand after cutting into small fragments incubated twice (30 minutes each)at 37° C. in F-10 medium containing 6% fetal calf serum and collagenase(2.5 mg/ml) (Boehringer, Mannheim GmbH, Germany). Fragments were thenwashed in Dulbecco's PBS, Ca² + and Mg² + free medium and mechanicallydissociated. Single cell suspension was planted onto 24-well (2×10⁵cells/well) culture plates. The cells were incubated in F-10 mediumsupplemented with 10% horse serum, 4% fetal calf serum and gentamycin(25 μg/ml), in a humidified atmosphere of 5% CO₂ and 95% air at 37° C.

After 3 days, the medium was removed and the cells washed twice withserum free F-10, then incubated with 1 ml of F-10 containing 0.1% BSAonly, or with added various concentrations of synthetic peptides.

After incubation for 2 h at 37° C., media were collected and storedfrozen at -20° C. until assayed for measurement of GH content.

In Vivo Experiments

Experimental Procedure

Between 09.00-10.00 h rats were anesthetized with ketamine (58 mg/kg,Inoketam, VIRBAC, Milano) and xilazine (12 mg/kg, Rompun, Bayer,Milano). Thirty minutes later, a blood sample (250 μg) was withdrawnfrom the exposed jugular vein, peptides were injected intravenously orsubcutaneously, and further blood samples were collected 10, 20 and 30minutes later.

Medium and plasma GH was measured by radioimmunoassay using materialssupplied by the NIADDK Bethesda, Md. Values were expressed in term ofNIASSK-rat-GH-RP-2 standard (potency 2 IU/ml), as ng/ml of medium orplasma.

The minimum detectable value of rat was 1.0 ng/ml; intra-assayvariability was 6%. To avoid inter-assay variation, samples from eachexperiment were assayed simultaneously. The results were as follows:

In Vitro Experiments

When pituitary cell monolayers were incubated for two hours withincreasing concentrations (10⁻⁸ to 10⁻⁶ M) of HEXARELIN and GHRP-6,stimulation of GH secretion over basal secretion was observed.Comparison of the GH secretion levels obtained after stimulation ofpituitary cell monolayers with GHRP-6 and HEXARELIN indicates that theiractivities were very similar, as shown in Table 1.

                  TABLE 1    ______________________________________    GH-RELEASING ACTIVITY OF GHRP-6 AND HEXARELIN           CONCENTRATION (M)    TREATMENT             0         10.sup.-8 10.sup.-7                                         10.sup.-6    ______________________________________    GHRP-6   484.2 ± 11.4                       544.2 ± 23.9                                 526.5 ± 19.0                                         510.0 ± 12.0    HEXARELIN             471.1 ± 31.3                       557.8 ± 15.9                                 589.1 ± 17.9                                         558.0 ± 19.1    ______________________________________

Pool of controls 492.5±12.4 GH (ng/well)

Values (ng/well) are the means +S.E.M. of 6 determinations per group.

Pituitary cell monolayers were incubated with peptides for 2 hours.

In Vivo Experiments

In anesthetized rats the administration of graded doses (150, 300 and600/μg/kg) of HEXARELIN elicited significant increases of plasma GHconcentrations 10 and 20 minutes after administration. Similar resultswere obtained after injection of the same doses of GHRP-6, as shown inTable 2.

                  TABLE 2    ______________________________________    COMPARISON OF THE GH-RELEASING ACTIVITY    OF GHRP-6 (A) AND HEXARELIN (B) ADMINISTERED I.V. IN    MALE RATS ANESTHETIZED WITH KETAMINEAND XILAZINE    TREAT- TIME (minutes)    MENT   0         10         20       30    ______________________________________    Control           19.8 ± 5.8(8)                      45.6 ± 7.5(8)                                 28.7 ± 4.8                                         32.2 ± 4.5(4)    A 150 μg/           30.2 ± 7.8(7)                     394.0 ± 31.0(7)                                139.3 ± 10.2(7)                                         42.5 ± 4.5(4)    kg    B 150 μg/           15.0 ± 2.9(8)                     412.7 ± 39.7(8)                                132.2 ± 12.7(8)                                         44.0 ± 6.0(4)    kg    A 300 μg/           21.9 ± 4.4(8)                     413.6 ± 21.5(8)                                162.7 ± 22.1(8)                                         38.2 ± 7.7(4)    kg    B 300 μg/           13.5 ± 2.0(7)                     438.5 ± 26.8(7)                                213.8 ± 34.2(7)                                         48.1 ± 8.8(3)    kg    A 600 μg/           21.2 ± 6.1(8)                     542.0 ± 38.0(8)                                195.5 ± 11.0(8)                                         64/0 ±    kg                                   11.9(4)    B 600 μg/           18.4 ± 4.3(8)                     478.3 ± 19.8(8)                                164.2 ± 13.2(8)                                         54.5 ±    kg                                   13.3(4)    ______________________________________

Values (ng/ml) are means±S.E.M.

Number of rats are shown in parentheses and refer to pooled data of 2-3experiments in which similar data were obtained.

These data are also illustrated in FIGS. 3-5.

Potency and time-course of the effects of the two peptides were almostsuperimposable. In anesthetized rats, subcutaneous administration ofHEXARELIN (150, 300 and 600 μg/kg) elicited a significant increase inplasma GH concentrations 10, 20 and 30 minutes after treatment. Asimilar profile of secretion was obtained after the administration ofGHRP-6 at the same dose levels, as shown in Table 3. In this instanceHEXARELIN appeared more effective than GHRP-6 at all the consideredtimes. In all, both peptides after subcutaneous administration eliciteda more prolonged stimulation of GH secretion although the maximum peaklevels were considerably lower than those reported after intravenousinjection.

                                      TABLE 3    __________________________________________________________________________    COMPARISON OF THE GH-RELEASING ACTIVITY OF    GHRP-6 (A) AND HEXARELIN (B) ADMINISTERED S.C.    IN MALE RATS ANESTHETIZED WITH KETAMINE AND XILAZINE           TIME (minutes)    TREATMENT           0      10     20      30    __________________________________________________________________________    Control           26.0 ± 8.0(11)                   22.0 ± 3.3(8)                          59.2 ± 8.1(11)                                  52.2 ± 5.5(11)    A 150 μg/kg           20.0 ± 5.0(8)                   63.1 ± 11.0(8)                         110.4 ± 18.0(8)                                  77.2 ± 14.0(8)    B 150 μg/kg           12.0 ± 4.0(7)                  107.1 ± 17.7(7)                         156.6 ± 18.7(7)                                  86.0 ± 18.9(7)    A 300 μg/kg           20.0 ± 6.0(8)                   63.9 ± 12.8(8)                         123.4 ± 14.6(8)                                  87.7 ± 11.8(8)    B 300 μg/kg           12.0 ± 4.0(7)                   80.6 ± 11.0(7)                         171.7 ± 22.0(7)                                 102.7 ± 17.0(7)    A 600 μg/kg           18.0 ± 4.0(10)                   93.1 ± 22.3(7)                         167.1 ± 14.7(10)                                 107.8 ± 9.5(10)    B 600 μg/kg           23.0 ± 6.0(10)                   90.7 ± 16.6(7)                         187.5 ± 15.4(10)                                 115.3 ± 19.1(10)    __________________________________________________________________________

Values (ng/ml) are means±S.E.M.

Number of rats are shown in parentheses and refer to pooled data of 2-3experiments in which similar data were obtained.

These data are also illustrated in FIGS. 6-8.

Example 11

Effect of HEXARELIN on GH Release in Pentobarbital-Anesthetized Rats

Male Sprague Dawley rats weighing 225-250 g were divided in groups offive animals each. Rats were anesthetized with Nembutal injectedintraperitoneally at 50 mg/kg, fifteen minutes prior to the first bloodwithdrawal taken over heparin by cardiac puncture (for determination ofbasal GH).

Subcutaneous injections of either HEXARELIN or GHRP-6 were givenimmediately after the first blood collection, and additional bloodsamples were collected 15 and 40 minutes later.

Measurement of rat GH was performed by a standard double antibodyradioimmunoassay with reagents supplied by the National Pituitary Agencyand the National Institute of Arthritis, Diabetes, and Digestive andKidney Diseases. The standards used were NIADDK-NIH-rGH-RP-2.Statistical data were obtained with the Student's t Test at asignificance level of 5%. Results are shown in Table 4.

                  TABLE 4    ______________________________________    COMPARATIVE EFFECT OF GHRP-6 AND HEXARELIN ON    GH RELEASE IN PENTOBARBITAL-ANESTHETIZED RATS    POST-DRUG PLASMA GH (ng/ml)    COMPOUND     0 min    15 min      40 min    ______________________________________    Saline s.c.  32 ± 15                           43 ± 21 128 ± 38    GHRP-6*    s.c.    50 μg/kg  57 ± 39                          262 ± 58  97 ± 44    25 μg/kg  41 ± 16                          222 ± 95 110 ± 47    HEXARELIN    s.c.    50 μg/kg  32 ± 16                          439 ± 69**                                       81 ± 13    25 μg/kg  56 ± 21                          388 ± 99*                                      100 ± 51    10 μg/kg  63 ± 58                           95 ± 44  88 ± 29    ______________________________________

Student's t test: * 1% P 5% ** 0.1% P 1%

Statistical values obtained for HEXARELIN at 50 μg/kg and 25 μg/kg arecompared to GHRP-6 at the same concentrations.

These data are also illustrated in FIGS. 9-11. Also, FIG. 12 illustratesthat HEXARELIN has greater hydrophobicity than GHRP-6.

Example 12

These two peptides were also tested for acute cardiovascular toxicity inthe rat.

    ______________________________________    HEXARELIN    GROUP      DOSE (mg/Kg) (*)                            NO. OF ANIMALS    ______________________________________    1          5            6    2          7.5          6    3          10           6    ______________________________________    GHRP-6    GROUP      DOSE (mg/Kg) ($)                            NO. OF ANIMALS    ______________________________________    4          2.5          6    5          5            6    6          7.5          6    ______________________________________     (*) The dose levels of HEXARELIN were established by the Sponsor on the     basis of a previous toxicity study.     ($) The dose levels of GHRP6 were established by the Sponsor on the basis     of literature data (Macia R.A. et al., Toxicol. Appl. Pharm. 194, 403-410     1990).

Dosages were calculated on the basis of the declared peptide content ineach product, as specified below:

1) HEXARELIN: peptide content 79%

2) GHRP-6: peptide content 64%

A single dose of HEXARELIN or GHRP-6 was administered to rats indifferent calendar dates in such a way that each group/dose should betreated in two subsequent days.

Initially 3 rats/group/compound, the first ones in numerical order, weretreated.

The treatment schedule was as follows:

    ______________________________________    DAYS: 1       2       3     4      5      6    ______________________________________    Groups          3 and 4 3 and 4 1 and 5                                1 and 5                                       2 and 6                                              2 and 6    Cages 5 and 7 6 and 8 1 and 9                                2 and 10                                       3 and 11                                              4 and 12    ______________________________________

For each compound, appropriate amounts of solutions in 0.9% NaCl forinjection were prepared just before treatment at the suitableconcentrations. The solutions were sterilized by filtration (Milliporefilter, pore size 0.22 μm). Owing to the type of the study (acute study)in which formulates were administered just after preparation, stabilitychecks were not performed. Concentration checks were also not performed.The volume of solution injected was maintained constant at 1 ml/Kg.

The intravenous injections were done as a single dose in one vein of thetail with an appropriately gauged sterile, disposable, plastic syringe.The injection rate was about 0.1 ml/sec.

Periodical observations were made up to 4 hours after treatment.Abnormality and mortality were recorded. Body weight was recorded onceduring pre-trial and on the administration day to calculate the volumesto be injected.

The mortality data obtained with the two products were:

    ______________________________________    Dose        Dead rats/Total N.sup.o of rats per group    ______________________________________    GHRP-6    2.5 mg/kg   0/6    5.0 mg/kg   1/6    7.5 mg/kg   2/6    HEXARELIN    5.0 mg/kg   0/6    7.5 mg/kg   1/6     10 mg/kg   2/6    ______________________________________

The results indicate that HEXARELIN shows the same lethality as GHRP-6but consistently at a higher dose, i.e., it is less toxic than GHRP-6,an unexpected finding particularly since HEXARELIN is more potentregarding its pharmacological activity.

Example 13

Stability of HEXARELIN Compared to GHRP6 After Irradiation in Solution

Solutions of Hexarelin and GHRP-6 in acetate buffer pH 5.4 (1 mg/ml w/v)were submitted to irradiation (Co 60) at doses varying from 0 to 1.6MRad with intervals of 2 MRad.

Subsequently, the samples were analyzed by RP-HPLC using 27%Acetonitrile in water as solvent, and the area of the peptide peak wasexamined.

The figure shows the variation of the percentage of residual materialaccording to the irradiation dose: ##EQU1##

Results are shown in FIG. 13.

Example 14

In this example, the neuroendocrine mechanism by which HEXARELIN andGHRP-6 mediate their actions has been compared. Although previousstudies have looked at the role of somatostatin in regulating the actionof GHRP-6 in culture and in stressed animals, this study observes therole of both somatostatin and GHRH in regulating the action of HEXARELINand GHRP-6 in conscious, freely-moving, nonstressed animals.

Sixty male rats were prepared with indwelling venous catheters underether anesthesia three days before experimentation. On the day ofexperimentation, all animals were given an iv heparin injection (100 IU;0630 h). At 0700 h, animals were treated with 0.5 ml of either normalserum (control-as), somatostatin antiserum (somatostatin-as; 0.25ml+0.25 ml saline), growth hormone-releasing hormone antiserum (GHRH-as;0.25 ml+0.25 ml saline), or both somatostatin antiserum and growthhormone-releasing hormone antiserum (0.25 ml somatostatin-as+0.25 mlGHRH-as). Blood sampling began 60 minutes after antiserum pretreatment,with blood samples collected every 20 minutes for three hours. After the180 minute sample (1100 h), animals were treated iv with 25 μg/kg ofeither Hexarelin or GHRP-6. Blood samples were then collected at 5, 10,15, 20, 30, 40, and 60 minutes after peptide treatment. All samples werecentrifuged immediately and the plasma frozen until assayed. The peak GHresponse to Hexarelin and GHRP-6 as well as the area under the responsecurves (AUCs) for the thirty minutes following peptide injection werecalculated. Data were subjected to repeated measures analysis ofvariance and are expressed as mean±SEM.

Factorial analysis of variance identified several main treatmenteffects.

A. Peptide Effects: The pooled results obtained from treatment witheither HEXARELIN or GHRP-6 suggest that, overall, HEXARELIN was moreeffective in eliciting a higher mean GH response as compared to GHRP-6,as shown in Table 5. GH AUC and peak GH responses were alsosignificantly higher.

B. Antisera Effects: Antisera pretreatment clearly demonstrated thatGHRH antiserum inhibited the GH response to both Hexarelin and GHRP-6.The mean GH response was significantly inhibited in GHRH antiserumpretreated rats as compared to animals which were not pretreated (Table6). The GH AUC and peak GH responses were significantly diminished.

                  TABLE 5    ______________________________________    Main Treatment Effects               Mean GH    GH AUC      Peak GH    Peptides   (ng/ml)    (ng/ml/30 min)                                      (ng/ml)    ______________________________________    Hexarelin  235 ± 21**                          7366 ± 912**                                      552 ± 59**    GHRP-6     131 ± 13                          4220 ± 665                                      293 ± 41    Antiserum (as)    control-as 241 ± 31                          7716 ± 1457                                      5#4 ± 97    somatostatin-as.               224 ± 23                          7011 ± 1603                                      516 ± 63    GHRH-as    116 ± 18.sup.##                          3771 ± 924.sup.##                                      288 ± 69.sup.#    somatostatin-as +                98 ± 14.sup.#                          3021 ± 565.sup.##                                      249 ± 46.sup.#    GHRH-as    ______________________________________     **(p < 0.01) significantly higher than GHRP6 treated animals.     .sup.# (p < 0.01), .sup.## (p < 0.05) significantly lower than control an     somatostatinas pretreated animals.

The responses of the individual treatment groups are also illustrated inFIGS. 15 to 19.

This Example investigated what role GHRH and somatostatin have in theneuroendocrine mechanism by which the GHRPs, HEXARELIN and GHRP-6,mediate their neuroendocrine effects. In vitro studies have suggestedthat the GHRPs exert an effect via a direct pituitary site of action.Here, however, the administration of HEXARELIN as well as GHRP-6 toconscious, freely-moving (non-stressed) animals, suggests that GHRH isintegrally involved in the mechanism by which HEXARELIN and GHPR-6mediate their GH-releasing effects in vivo. This corroborates an earlierstudy in acutely-treated, stressed animals where passive immunization ofendoqenous GHRH resulted in a diminished plasma GH diminished plasma GHresponse to GHRP-6.

It has previously been suggested that somatostatin is involved in themechanism by which GHRP-6 mediates its neuroendocrine effects. This wasan acute study, however, conducted in a fashion known to induce stress,and thus, increase somatostatin tone in rats. In contrast, the resultsof the present study suggest minimal somatostatin involvement. We findthese results surprising, both in light of the previous study and sincewe have found somatostatin to be involved in most GH-releasingmechanisms previously examined. The apparent disparity in resultsbetween the two studies may be accounted for by the fact that weperformed our study in non-stressed, conscious, freely-moving rats. Insuch non-stressed animals, somatostatin tone is variable: low during aGH peak, or high may underestimate the importance of somatostatin inthis mechanism. For these reasons, we are hesitant to exclude theinvolvement of somatostatin at this time and feel that further analysisof somatostatin's involvement is warranted.

Example 15

The effects of HEXARELIN on growth hormone secretion in young (20-30years old) healthy male volunteers were measured after theadministration of various dosages. The results shown in FIGS. 18-20demonstrate that the peptide is effective in vivo as well as in vitro.

Example 16

The peptide of Example 3 was formulated in a polymeric PLGA implant inthe form of rods which were about 1 cm long and 1 mm in diameter. Theseimplants contained a loading of either 20 or 25% of the peptide (anamount of 7 or 10 mg), and were inserted subcutaneously into in maleBeagle dogs which weighed between 10 and 12 kg. After an initialflare-up, plasma testosterone fell below castration levels afterapproximately 10 days, and was maintained for approximately 180 days.The absence of response after a stimulation by i.v. administration ofthe peptide at day 145 indicates down-regulation of the pituitaryreceptors. No clinical side effects were observed during this study.

Although the aforementioned examples of the present invention disclosespecific embodiments thereof, it is believed that the substitution of anD-2-alkylTryptophan in bioactive peptides which contain at least oneTryptophan residue will yield bioactive peptides providing theadvantages and benefits discussed above.

The incorporation of a D-2-alkylTryptophan in bioactive peptides asdescribed above provides a method for prolonging and preserving theactivity of such peptides. When analogous bioactive peptides notsubstituted with an D-2-alkylTryptophan are exposed to variousprocessing conditions and substances, the activity of such peptides maybe adversely effected. Sterilizing procedures used in the pharmaceuticalindustry may expose bioactive compounds to ionizing radiation. Suchradiation may effect the formation of reactive radicals. Tryptophancontaining peptides are particularly susceptible to attack by suchradicals and such attack may render the peptide ineffective, or possiblytoxic.

Furthermore, various formulating compounds, such aspolylactic-polyglycolic acid (PLGA) polymers may contain active, oractivated groups which may also attack Tryptophan containing bioactivepeptides. The present invention provides a method for protecting atryptophan containing bioactive peptide from these manufacturing hazardswhile also increasing the peptides resistance to oxidative degradationafter formulation is complete. It is believed that the presence of thealkyl group at the number 2 position of the Tryptophan increases thestability of the pyrrole ring wherein attack by reactive radicals andactive or activated groups occurs.

Example 17

Making use of the solid-phase peptide synthesis technique as describedin "Solid phase peptide synthesis" by E. Atherton and R. C. Sheppard,IRL Press, Oxford University Press, 1984, using fluorenylmethoxycarbonyl(Fmoc) as the protecting group, the peptide:

    GAB-D-2-Mrp-D-2-Mrp-Phe-Lys-NH.sub.2,

was prepared, wherein Mrp is 2-methyltryptophan, M.W. 779.9, found778.4; purity (HPLC) 98.0%

Example 18

Analogously to Example 17, the following peptide was prepared:GAB-D-2-Mrp-D-2-Mrp-2-Mrp-Lys-NH₂, wherein Mrp is 2-methyltryptophan,M.W. 830.8, found 831.3; purity (HPLC) 98.0%.

Example 19

Analogously to Example 17, the following peptide was prepared:Aib-D-2-Mrp-D-2-Mrp-NH₂,

wherein Mrp is 2-methyltryptophan, M.W. 502.6, found 503.3; purity(HPLC) 99.0%.

Example 20

Analogously to Example 17, the following peptide was prepared:Aib-D-2-Mrp-2-Mrp-NH₂,

wherein Mrp is 2-methyltryptophan, M.W. 502.6, found 503.3; purity(HPLC) 99.0%.

Example 21

Analogously to Example 17, the following peptide was prepared:Aib-D-Ser(Bzl)-D-Mrp-NH₂,

wherein Mrp is 2-methyltryptophan, M.W. 479.6, found 480.5; purity(HPLC) 99.0%.

Example 22

Analogously to Example 17, the following peptide was prepared:GAB-D-2-Mrp-D-βNal-Phe-Lys-NH₂,

wherein 2-Mrp is 2-methyltryptophan, M.W. 774.8, found 775: purity(HPLC) 99.0%.

Example 23

Analogously to Example 17, the following peptide was prepared:GAB-D-2-Mrp-D-2-Mrp-D-2-Mrp-Lys-NH₂,

wherein 2-Mrp is 2-methyltryptophan, M.W. 830.8, found 831.5: purity(HPLC) 99.0%.

Example 24

Analogously to Example 17, the following peptide was prepared:D-2-Mrp-D-2-Mrp-2-Mrp-NH₂,

wherein 2-Mrp is 2-methyltryptophan, M.W. 617.7, found 618.3: purity(HPLC) 99.0%.

Example 25

Analogously to Example 17, the following peptide was prepared:D-2-Mrp-2-Mrp-NH₂,

wherein 2-Mrp is 2-methyltryptophan, M.W. 417.5, found 418.3: purity(HPLC) 99.0%.

Example 26

Analogously to Example 17, the following peptide was prepared:GAB-D-2-Mrp-2-Mrp-NH₂,

wherein 2-Mrp is 2-methyltryptophan, M.W. 502.6, found 503.2: purity(HPLC) 99.0%.

Example 27

Biological Activity

In vivo activity of these compounds was determined in ten day-rats,which were subcutaneously injected (s.c.) with a dose of 300 μg/kg orwith different doses in dose-response studies, according to methodsdescribed in detail by R. Deghenghi et al, Life Sciences, 54, 1321,(1994). The results are provided in Table 6 below. The released GH wasmeasured after 15 minutes from the treatment.

                  TABLE 6    ______________________________________                              released GH    Peptide of example                   Dose μg/kg s.c.                              (ng/ml)    ______________________________________    17             300        155.4 ∓ 19.7    18             300        165.4 ∓ 18.5    19             300        174.2 ∓ 25.9    20             300         64.2 ∓ 12.6    21             1.2 mg/kg   59.4 ∓ 12.3    22             300        145.7 ∓ 9.0    23             300        91.2 ∓ 9.0    24             300        26.3 ∓ 5.0    25             300        27.0 ∓ 4.8    26             300        36.0 ∓ 9.5    Controls       --         15.7 ∓ 6.7    ______________________________________

The data shows that the peptides of Examples 17, 18, 19 and 23 are themost active.

Example 28

A capsule containing 20 mg of the peptide of Example 18 was orallyadministered to 5 healthy subjects (3 men and 2 women, ages 30 to 66)and serum growth hormone and cortisol levels were measured at varioustimes after administration. Results are shown in FIGS. 21A and 21B.Surprisingly, growth hormone levels were increased without cortisolstimulation.

Examples 29-31

The peptides of Examples 18, 19 and 23 were selected for further studyand were tested in vivo in adult dogs. The peptides were administeredorally at a dosage of 1 mg/Kg. body weight. Results are shown in FIGS.22-24.

Example 32-33

The peptides of Examples 19 and 23 were further studied for inhibitionof ¹²⁵ I-Tyr-Ala-HEXARELIN binding to human tissue.

Tissue membranes (0.1 mg protein) obtained from the heart andhypothalamus of two different adult subjects were incubated intriplicate with a subsaturating concentration (34 pM, about 48,000 cpm,for heart tissue and 42 pM, about 60,000 cpm for hypothalamus tissue) of¹²⁵ I-Tyr-Ala-HEXARELIN for 40 min at 0° C. in the absence and in thepresence of increasing concentrations of the indicated unlabelledpeptides. The value in parentheses represents the % of inhibition of ¹²⁵I-Tyr-Ala-HEXARELIN specifically bound. Results are shown in Tables 7and 8 below.

                                      TABLE 7    __________________________________________________________________________    Human heart tissue    peptide concentration              HEXARELIN Example 19                                  Example 23    (nM)      subject 1                   subject 2                        subject 1                             subject 2                                  subject 1                                       subject 2    __________________________________________________________________________    0         21.0 (0)                   23.9 (0)                        25.1 (0)                             24.6 (0)                                  20.3 (0)                                       24.0 (0)    0.1       20.1 (4)                   22.8 (5)                        25.1 (0)                             24.5 (0)                                  20.1 (1)                                       23.8 (1)    1         16.9 (20)                   17.3 (28)                        24.9 (1)                             24.5 (0)                                  19.8 (3)                                       23.5 (2)    10        10.4 (51)                   10.5 (56)                        24.9 (1)                             24.0 (3)                                  16.5 (19)                                       19.2 (20)    100        1.7 (92)                    2.4 (90)                        24.7 (2)                             24.1 (2)                                  10.2 (50)                                       11.2 (53)    1000        0 (100)                     0 (100)                        24.4 (3)                             24.0 (3)                                   4.0 (80)                                        4.0 (83)    IC.sub.50 value (nM)              10.6  8.3 inactive                             inactive                                  62.7 57    mean IC.sub.50 value (nM)              9.5       --        59.8    comparative IC.sub.50 value              1         --        6    (HEXARELIN = 1)    __________________________________________________________________________

                                      TABLE 8    __________________________________________________________________________    Human hypothalamus tissue    peptide concentration              HEXARELIN Example 19                                  Example 23    (nM)      subject 1                   subject 2                        subject 1                             subject 2                                  subject 1                                       subject 2    __________________________________________________________________________    0         11.2 (0)                   9.5 (0)                        11.0 (0)                             10.3 (0)                                  10.2 (0)                                       10.8 (0)    0.1       10.7 (3)                   9.1 (4)                         9.8 (11)                              9.0 (13)                                   9.1 (11)                                       10.0 (7)    1          9.6 (16)                   7.7 (19)                         8.3 (25)                              7.0 (32)                                   8.8 (14)                                        8.9 (18)    10         5.2 (51)                   3.1 (67)                         3.9 (65)                              3.6 (75)                                   3.8 (63)                                        3.3 (69)    100        1.2 (89)                    0.7 (93)                         1.0 (91)                              0.2 (98)                                   2.1 (79)                                        1.9 (82)    1000        0 (100)                     0 (100)                          0 (100)                              0.1 (99)                                   0.3 (97)                                         0 (100)    IC.sub.50 value (nM)               8.9 5.0   6.0  3.6  6.2  4.5    mean IC.sub.50 value (nM)              7.0       4.8       5.4    comparative IC.sub.50 value              1         0.7       0.8    (HEXARELIN = 1)    __________________________________________________________________________

The specific binding of ¹²⁵ I-Tyr-Ala-HEXARELIN/0.1 mg membrane proteinis expressed as a percent of total radioactivity added. AlthoughHEXARELIN is useful, the results show that the peptide of Example 19exhibits potent binding to hypothalamus tissue with no binding to hearttissue, while the peptide of Example 23 also exhibits strong binding tohypothalamus tissue with only moderate binding to heart tissue.

Example 34

Synthesis of GAB-D-Mrp-D-Trp-Phe-Lys-NH₂

The synthesis of the title peptide was carried out by solid-phase with9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids involvingresin preparation and assembly in a reactor column according to one ofseveral methods known to those skilled in the art, as exemplified in"Solid phase peptide synthesis" by E. Atherton and R. C. Sheppard, IRLpress at Oxford University press, 1989. The protected amino acids areFmoc-Lys(Fmoc)-Opfp (Opfp=pentafluorophenyl ester), Fmoc-Phe-Opfp,Fmoc-D-Trp-Opfp, Fmoc-D-2-Me-Trp-Opfp and Fmoc-GAB-Opfp(GAB=gamma-aminobutyryl). Alternatively, the use of Castro's reagents,benzotriazolyloxy-tris(dimethylamino) phosphonium (hexafluorophosphate)BOP and the pyridinium analog of BOP (PyBOP) (cfr. Le Nguyen and Castro(1988) in Peptide Chemistry 1987, p. 231-238; Protein ResearchFoundation Osaka; and Tetrahedron Letters 31, 205 (1990)) can be usedadvantageously as direct coupling reagents.

After cleavage and isolation, the title peptide was purified as itsacetate salt. Purity (HPLC): 98%, MW (M+H⁺)=764.3 (theoretical=763.9).

Example 35

The following peptides were prepared according to the proceduresdescribed in Example 34, isolated as their TFA (triflouroacetate) saltsand whenever needed, purified as their acetate salts:

INIP-D-Mrp-D-Trp-Phe-Lys-NH₂, purity (HPLC)=99.0%, MW (M+H⁺ =790.4;theoretical=790.0);

INIP-D-Mrp-D-β-Nal-Phe-Lys-NH₂, purity (HPLC)=96.5%, MW (M+H⁺ =801.4;theoretical=801.0);

IMA-D-Mrp-D-Trp-Phe-Lys-NH₂, purity (HPLC)=99.2%, MW (M+H⁺ =786.5;theoretical=786.8);

IMA-D-Mrp-D-β-Nal-Phe-Lys-NH₂, purity (HPLC)=97.3%, MW (M+H⁺ =798.3;theoretical=797.9);

GAB-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ;

GAB-D-Mrp-D-β-Nal-NH₂ ;

4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-Trp-Phe-Lys-NH², purity(HPLC)=99.0%, MW (M+H⁺ =818.5; theoretical=818.0);

4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-β-Nal-Phe-Lys-NH₂, purity(HPLC)=96.9%, MW (M+H⁺ =829.5; theoretical=829.1);

D-Ala-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂, purity (HPLC)=99.3%, MW (M+H⁺ =821.3;theoretical=821.9);

D-Thr-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂, purity (HPLC)=98.0%, MW (M+H⁺ =851.5;theoretical=852.0);

His-D-Mrp-Ala-Phe-D-Trp-Lys-NH₂, purity (HPLC)=22 98.6%, MW (M+H⁺=887.4; theoretical=888.0);

Tyr-His-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂, purity (HPLC)=96.8%, MW (M+H⁺=1050.2; theoretical=1050.2);

His-D-Mrp-Ala-Trp-NH₂, purity (HPLC)=99.8%, MW (M+H⁺ =612.3;theoretical=612.7);

D-Thr-D-Mrp-Ala-Trp-NH₂, purity (HPLC)=97.5%, MW (M+H⁺ =576.5;theoretical=576.6);

His-D-Mrp-Ala-Trp-D-Phe-Lys-Thr-NH₂ ;

His-D-Mrp-Ala-Trp-D-Phe-Lys-D-Thr-NH₂ ;

D-Thr-His-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂ ; and

imidazolylacetyl-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂,

wherein Mrp is 2-methyltryptophan, and INIP, IMA and GAB are as definedabove.

Example 36

Synthesis of GAB-D-Mrp-D-β-Nal-OC₂ H₅.

The peptide GAB-D-Mrp-D-β-Nal-OC₂ H₅, bearing an ethyl ester in theC-terminal position, was synthesized via solution-phase synthesisaccording to conventional methods such as those described in of Bodanskyet al., Peptide Synthesis, 2nd edition, John Wiley & Sons, New York, N.Y. 1976 and Jones, The Chemical Synthesis of Peptides (Clarendon Press,Oxford, 1994), wherein the starting material used in the synthesis ofthe title compound was D-β-naphthylalanine ethyl ester.

Example 37

Biological Activity

In vivo activity of these compounds was determined in ten day-rats,which were subcutaneously injected (s.c.) with a dose of 300 μg/kg orwith different doses in dose-response studies, according to theprocedure as described in Deghenghi et. al, Life Sciences 54, 1321(1994). The results are shown in Table 9, below. The released GH wasmeasured 15 minutes following compound administration.

                                      TABLE 9    __________________________________________________________________________                            Dose GH control                                       GH released    Peptide                 μg/kg s.c.                                 (ng/ml)                                       (ng/ml)    __________________________________________________________________________    His-D-Mrp--Ala--Trp-D-Phe--Lys--Thr--NH.sub.2                            300  31 ± 8                                       176 ± 20    His-D-Mrp--Ala--Trp-D-Phe--Lys--NH.sub.2                            300  14.7 ± 1.9                                       104.2 ± 13.1    His-D-Mrp--Ala--Trp-D-Phe--Lys-D-Thr--NH.sub.2                            300  31 ± 8                                       169 ± 27    D-Thr-D-Mrp--Ma--Trp-D-Phe--Lys--NH.sub.2                            300  31 ± 8                                       266 ± 20    D-Thr--His-D-Mrp--Ala--Trp-D-Phe--Lys--NH.sub.2                            300  31 ± 8                                        86 ± 19    D-Ala-D-Mrp--Ala--Trp-D-Phe--Lys--NH.sub.2                             40  34 ± 1                                       200 ± 20    D-Ala-D-Mrp--Ala--Trp-D-Phe--Lys--NH.sub.2                            320  34 ± 1                                       251 ± 32    His-D-Mrp--Ala--Trp--NH.sub.2                            5000  69 ± 14                                       124 ± 37    imidazolylacetyl-D-Mrp--Ala--Trp-D-Phe--Lys--NH.sub.2                            300  20 ± 3                                       159 ± 27    imidazolylacetyl-D-Mrp-D-Trp--Phe--Lys--NH.sub.2                            300  14.7 ± 1.9                                       60.3 ± 8.1    imidazolylactyl-D-Mrp-D-β-Nal--Phe--Lys--NH.sub.2                            300  14.7 ± 1.9                                        56.0 ± 12.4    INIP-D-Mrp-D-Trp--Phe--Lys--NH.sub.2                            300  15    155    INIP-D-Mrp-D-Trp--Phe--Lys--NH.sub.2                            300  14.7 ± 1.9                                       119.5 ± 18.6    INIP-D-Mrp-D-β-Nal--Phe--Lys--NH.sub.2                            300  15    150    INIP-D-Mrp-D-β-Nal--Phe--Lys--NH.sub.2                            300  14.7 ± 1.9                                       125.9 ± 13.0    4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-                            300  14.7 ± 1.9                                       111.8 ± 24.6    Trp--Phe--Lys--NH.sub.2    GAB-D-Mrp-D-Trp--Phe--Lys--NH.sub.2                            300  10    110    GAB-D-Mrp-D-Trp--Phe--Lys--NH.sub.2                            300  14.7 ± 1.9                                       172.8 ± 15.8    GAB-D-Mrp-D-β-Nal--Phe--Lys--NH.sub.2                            300  14.7 ± 1.9                                       198.0 ± 13.2    (GHRP-2) - reference    300  10    98.6    (GHRP-2) - reference    300  14.7 ± 1.9                                       154.4 ± 18.5    __________________________________________________________________________

The GHRP-2 (reference standard) has the structureD-Ala-D-β-Nal-Ala-Trp-D-Phe-Lys-NH₂ (Chen and Clarke, J. Neuroend. 7,179 (1995).

In vitro measurements of adenylcyclase activity were determined inanterior pituitary gland cells from rats weighing 150 g and showed a 30%increase compared with the baseline with EC₅₀ =0.23 nM for the peptideD-Ala-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂, whereas GHRP-6(His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂) resulted inactive.

From the foregoing examples, it is evident that the incorporation of aD-2-methyl-tryptophan residUe in the peptides of the invention result inincreased activity ranging from 30-40 ng/ml to over 200 ng/ml. Also,this increased activity is tissue selective for certain peptides. Forthese reasons, the peptides of this invention have greaterpharmacological utility than the corresponding peptides that do notcontain D-2-Mrp.

While it is apparent that the invention herein disclosed is wellcalculated to fulfill the objects above stated, it will be appreciatedthat numerous embodiments and modification may be devised by thoseskilled in the art, and it is intended that the appended claims coverall such modification and embodiments as fall within the true spirit andscope of the present invention.

What is claimed is:
 1. A method for enhancing the growth hormone releasewhich comprises formulating a composition comprising a peptide having anamino acid sequence which includes at least one D-2-alkylTryptophan thathas a lower alkyl group substituted at the number 2 position, andadministering a therapeutically effective amount of the composition toan animal to obtain enhanced growth hormone release compared to therelease evoked by peptides that have conventional Tryptophan in place ofthe D-2-alkylTryptophan.
 2. The method according to claim 1, wherein theanimal is a human.
 3. The method according to claim 1, wherein thepeptide is administered intravenously in an amount of about 0.1 μg toabout 10 μg of total peptide per kg of body weight.
 4. The method ofclaim 1 wherein the peptide sequence contains between 2 and 10 aminoacids, wherein at least one amino acid is the D-2-alkylTryptophan. 5.The method of claim 4 wherein the peptide sequence contains at least twoamino acids which are D-2-alkylTryptophan.
 6. The method of claim 5,wherein the two D-2-alkylTryptophans are in adjacent positions in thepeptide sequence.
 7. The method of claim 1, wherein the lower alkylgroup is methyl, ethyl, propyl or isopropyl.
 8. The method of claim 1,wherein the lower alkyl group is a methyl group.
 9. The method of claim1, wherein the peptide has one of the following formulae:

    A--D--X--Z--B                                              (I)

    E--D--Mrp--(Ala).sub.n --F--G                              (II)

    J--D--X--Mrp--NH.sub.2                                     (III)

wherein A is hydrogen, 2-aminoisobutyryl, or 4-aminobutyryl; D standsfor the dextro isomer, X is a 2-alkyltryptophan of formula (IV):##STR4## wherein R is hydrogen, CHO, SO₂ CH₃, mesitylene-2-sulfonyl, PO₃(CH₃)₂, PO₃ H₂, wherein R₁ is a C₁ -C₃ alkyl group (e.g., methyl, ethyl,propyl or isopropyl), or X is a residue of protected serine, Ser (Y),wherein Y can be benzyl, p-chlorobenzyl, 4-methoxybenzyl,2,4,6-trimethoxybenzyl, or t-butyl, Z is D-Mrp, D-βNal or Mrp; Mrp is2-alkyl-Tryptophan; B is NR₂ R₃, wherein R₂ and R₃, which can be thesame or different, are hydrogen, a C₁ -C₃ alkyl group, an OR₄ group,wherein R₄ is hydrogen, a C₁ -C₃ alkyl, or a C-Lys-NH₂ group, wherein Cis Phe, Mrp or D-Mrp; E is any natural L-amino acid or its D-isomer,imidazolylacetyl, isonipecotinyl, 4-aminobutyryl,4-(aminomethyl)cyclohexanecarbonyl, Glu-Tyr-Ala-His, Tyr-Ala-His,Tyr-His, D-Thr-His, D-Ala, D-Thr, Tyr and Gly; n is 0 or 1; F isselected from the group consisting of Trp, D-Trp, Phe and D-β-Nal; G isselected from the group consisting of NH₂,D-Phe-Lys-NH₂, Phe-Lys-NH₂,D-Trp-Lys-NH₂, D-Phe-Lys-Thr-NH₂, D-Phe-Lys-D-Thr-NH₂ and an O--C₁ -C₃alkyl group, with the proviso that E is not His when F is L-Trp or D-Trpand when G is D-Phe-Lys-NH₂ ; and J is hydrogen, GAB or D-Mrp;or os apharmaceutically acceptable addition salt thereof.
 10. The method ofclaim 1, wherein the peptide isGAB-D-Mrp-D-Mrp-Phe-Lys-NH₂ ;GAB-D-Mrp-D-Mrp-Mrp-Lys-NH₂ ; Aib-D-Mrp-D-Mrp-NH₂ ; Aib-D-Mrp-Mrp-NH₂ ;Aib-D-Ser(Bzl)-D-Mrp-NH₂ ; GAB-D-Mrp-D-βNal-Phe-Lys-NH₂ ;GAB-D-Mrp-D-Mrp-D-Mrp-Lys-NH₂ ; D-Mrp-D-Mrp-Mrp-NH₂ ; GAB-D-Mrp-Mrp-NH₂; D-Mrp-Mrp-NH₂ INIP-D-Mrp-D-Trp-Phe-Lys-NH₂ ;INIP-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ; IMA-D-Mrp-D-Trp-Phe-Lys-NH₂ ;IMA-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ; GAB-D-Mrp-D-Trp-Phe-Lys-NH₂ ;GAB-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ; GAB-D-Mrp-D-β-Nal-NH₂ ;imidazolylacetyl-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂ ;imidazolylacetyl-D-Mrp-D-Trp-Phe-Lys-NH₂ ;imidazolylacetyl-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ;4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-Trp-Phe-Lys-NH₂ ;4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-β-Nal-Phe-Lys-NH₂ ;D-Ala-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂ ; D-Thr-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂ ;His-D-Mrp-Ala-Phe-D-Trp-Lys-NH₂ ; His-D-Mrp-Ala-Trp-D-Phe-Lys-Thr-NH₂ ;His-D-Mrp-Ala-Trp-D-Phe-Lys-D-Thr-NH₂ ;Tyr-His-D-Mrp-Ala-Trp-D-Phe-LysNH₂ ; His-D-Mrp-Ala-TrpNH₂ ;D-Thr-His-D-Mrp-Ala-Trp-D-Phe-Lys-NH₂ ; D-Thr-D-Mrp-Ala-TrpNH₂ ; orGAB-D-Mrp-D-β-Nal-OC₂ H₅ and pharmaceutically acceptable salts thereof,wherein Mrp is 2-methyltryptophan.